Research Progress on Strategies for Improving the Enzyme Properties of Bacteriophage Endolysins

Abstract

lysins' ' [4].According to the Gram-staining of host bacteria, bacteriophage endolysins are divided into two groups.One could be called GP-Lysins which are produced by bacteriophages of Gram-positive bacteria (GP-phages).The other one could be called GN-Lysins which are produced by bacteriophages of Gram-negative bacteria (GNphages).GP-Lysins usually have a modular structure.As shown in Fig. 1A, GP-Lysins contain two domains, namely the N-terminal catalytic domain (EAD) and the C-terminal cell wall binding domain (CBD), which are connected by a short peptide.The EAD is capable of acting on most chemical bonds of the PG network in the bacterial cell wall to cause bacterial lysis [1], while the CBD is responsible for targeting the endolysin to the substrate and conferring specificity for recognizing host cells.The high specificity of lysins offers it an advantage over conventional antibiotics as endolysins do not disturb the normal microflora.Typically, these is a flexible interdomain linker sequence between the EAD and the CBD [5].Usually, endolysins have only one EAD and one CBD, but some lysins were found to have more than one CBD or EAD arranged in different order [6][7][8].As shown in Fig. 1B, GN-lysins usually only have one globular structure with a single EAD [9, 10].Gramnegative bacteria have an outer membrane (OM) composed of lipopolysaccharide (LPS) compared to Grampositive bacteria.The OM of Gram-negative bacteria effectively prevents GN-lysins from acting on the cell wall externally.To date, only a few GN-lysins are able to lyse Gram-negative bacteria without the help of OM permeants.These GN-lysins contain amphipathic helical structures or carry positively charged groups which confer the ability to penetrate or disrupt the bacterial OM, thereby accessing and degrading the PG layer and ultimately leading to bacterial lysis and death [11,12].For example, the -helical structure formed by the Cterminus of lysin AcLys enables it to penetrate the OM.Additionally, the positively charged groups present in the C-terminus enhance its ability to penetrate the OM [11,13].However, there are also a few of GN-Lysins owning a modular structure with a CBD at the N-terminus and a EAD at the C-terminus [14,15].These GN-Lysins obtain high lytic activities towards Gram-negative bacteria due to the presence of CBD which helps the lysins get close to Bacterial resistance to commonly used antibiotics is one of the major challenges to be solved today.Bacteriophage endolysins (Lysins) have become a hot research topic as a new class of antibacterial agents.They have promising applications in bacterial infection prevention and control in multiple fields, such as livestock and poultry farming, food safety, clinical medicine and pathogen detection.However, many phage endolysins display low bactericidal activities, short half-life and narrow lytic spectrums.Therefore, some methods have been used to improve the enzyme properties (bactericidal activity, lysis spectrum, stability and targeting the substrate, etc) of bacteriophage endolysins, including deletion or addition of domains, DNA mutagenesis, chimerization of domains, fusion to the membrane-penetrating peptides, fusion with domains targeting outer membrane transport systems, encapsulation, the usage of outer membrane permeabilizers.In this review, research progress on the strategies for improving their enzyme properties are systematically presented, with a view to provide references for the development of lysins with excellent performances.

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